Genetically controlling VACUOLAR PHOSPHATE TRANSPORTER 1 contributes to low-phosphorus seeds in Arabidopsis

ABSTRACT Phosphorus (P) is an indispensable nutrient for seed germination, but the seeds always store excessive P over demand. High-P seeds of feeding crops lead to environmental and nutrition issues, because phytic acid (PA), the major form of P in seeds, cannot be digested by mono-gastric animals. Therefore, reduction of P level in seeds has become an imperative task in agriculture. Our study here suggested that both VPT1 and VPT3, two vacuolar phosphate transporters responsible for vacuolar Pi sequestration, were downregulated in leaves during the flowering stage, which led to less Pi accumulated in leaves and more Pi allocated to reproductive organs, and thus high-P containing seeds. To reduce the total P content in seeds, we genetically regulated VPT1 during the flowering stage and found that overexpression of VPT1 in leaves could reduce P content in seeds without affecting the production and seed vigor. Therefore, our finding provides a potential strategy to reduce the P level of the seeds to prevent the nutrition over-accumulation pollution.


Introduction
Phosphorus (P) is an essential macronutrient for all bioorganisms, while phosphate (Pi) is the major form of P that can be utilized by plants. Because of its pivotal roles in numerous primary bio-metabolisms, such as formation of nucleic acids, lipid synthesis, protein phosphorylation, etc., Pi is indispensable for plant growth and development. Pi transporters are known as the functional components that facilitate Pi transportation and contribute to fine-tuning of Pi homeostasis in plants 1,2 . So far, vacuole is defined as the biggest Pi pool that is critical for maintaining Pi balance in plant cells. People have made big progresses in identification of the long-sought vacuolar Pi transporters, recently. VPT1 (Vacuolar Phosphate Transporter 1, also named PHT5;1) family members were found to be the vacuolar Pi influx transporters that are responsible for vacuolar Pi storage under Pi sufficient conditions 3,4 , while VPE (Vacuolar Phosphate Efflux) family members were identified as the vacuolar Pi efflux transporters that contribute to vacuolar Pi remobilization in response to Pi deficiency 5 . These two types of vacuolar transporters are vital for Pi homeostasis in plants.
With the assistance of multiple transporters, Pi is transported into or out-of various organelles, cells and tissues for acclimating to the physiological demand of plants. Especially, in different development stages, the plant adopts different policies for Pi distribution in tissues. During the flowering stage, physiological changes and expression of specific genes would cater to reproduction. Pi is essential for reproduction, and sufficient available Pi contributes to the transformation from vegetative growth to flowering in plants. Additionally, from flowering to seed maturation, large amounts of Pi are allocated to the reproductive organs for synthesis of phytic acid (PA, or InsP 6 ) which is referred as the major stored form of P in seeds [6][7][8] . Although PA is required for germination of seeds, most crop seeds contain much higher PA beyond the quantity of demand 9 . As many crop seeds such as corn and sorghum seeds are often used for animal feed, this high level of PA in seeds leads to severe water eutrophication. It has been known that mono-gastric animals cannot digest PA and most of PA in feed is excreted into lakes, rivers and the groundwater 10 . Moreover, PA could easily chelate Fe, Cu and Zn that are essential elements for the activities of digestive enzymes; thus, high level of PA contributes to malnutrition of the animals 10 . Therefore, PA content or P accumulation in seeds should be reduced to solve these environmental and nutritional issues. However, suppressing the allocation of P to the seeds by using efficient genetic target is largely unexplored.
Our previous work suggested that VPT1 family members were essential for maintaining the systemic Pi balance during flowering. Loss of function of VPT1 and VPT3 would affect the long-distance Pi transport and result in toxic level of Pi allocated to the floral organ rather than stored in vacuole of leaves 11 . Therefore, genetically controlling the VPT proteins may reduce the Pi level in reproductive organs and seeds. In this study, we uncovered that the expression of VPT1 and VPT3 was downregulated in leaves during flowering stage; this regulation may contribute to more Pi allocated to reproductive organs for enhancement of the PA synthesis in seeds. Through a dexamethasone-inducible expression system, we found that overexpression of VPT1 in leaves resulted in less Pi in the stem xylem sap and thus reduced P accumulation in the floral organ and seeds. Our finding should provide available genetic tools for generating the low-P grain crops.

Vacuolar Pi transporter genes are transcriptionally regulated for controlling Pi status in the reproductive organ
Pi transport in plants is regulated according to nutrientdemanding policies during various development stages. Especially for the flowering, one of the most energy and nutrient consuming development stage, Pi transport should be strictly controlled for reproduction. To detect the Pi distribution pattern in a flowering Arabidopsis plant, we collected different tissues, including roots, rosette leaves, stems, stem leaves and floral organs for Pi content measurements. As shown in Figure 1, the rosette leaves contained more Pi than roots, while the Pi content of stem leaves was higher than that of rosette leaves. The stems contained the lowest Pi, while the Pi content of floral organs was highest among all the tissues.
During flowering stage, root-to-flower Pi transport should be precisely regulated to maintain a proper Pi status in the reproductive organ, which contributes to a high quality of production. Our previous study suggested that vacuolar phosphate transporters were very critical for long-distance Pi transport in Arabidopsis, and deletion of two vacuolar influx Pi transporters (VPT1 and VPT3) would result in more Pi delivered into the floral organ and finally affect seed set 11 . To test how VPT1 and VPT3 are regulated in different development stages, we conducted qPCR to explore the expression patterns of these two important Pi transporter genes. The data shown in Figure 2a illustrated that before flowering, VPT1 and VPT3 were expressed highly in leaves, whereas a dramatically transcriptional down-regulation occurred when seedlings were full flowering. However, in roots, the transcripts of VPT1 and VPT3 are not significantly regulated in different development stages (Figure 2b). The data of GUS staining assay Figure 1. Pi distribution pattern in various tissues. Different tissues including roots, rosette leaf, stem, stem leaf and floral organ were collected from the flowering WT seedlings for Pi content measurements. Different letters above each bar indicate statistically significant differences between various tissues (P < 0.05, Tukey's honestly significant difference test). Error bars indicate ± SD; n = 4 biological replicates, each with three technical replicates. were consistent with the qPCR results (Figure 2c, d). Then, we collected rosette leaves and roots from plants that were in the "before flowering" and the "full flowering" stages separately and measured their Pi contents. The data suggested that Pi contents of roots were not obviously regulated, whereas the Pi content was significantly decreased in "full flowering" leaves than in "before flowering" leaves ( Figure 2e). As previous work indicated that loss of function of VPT proteins results in excess of Pi allocated to the floral organs rather than stored in the vacuoles of leaves, we proposed that VPT genes were downregulated for more Pi allocated to the floral organ for meeting the physiological demand of the reproductive organs. During seed maturation, the longdistance transported Pi would be involved in the synthesis of PA that is the major storage form of phosphorus in seeds 12 . Therefore, the more Pi is transported into reproductive organ, the higher PA level in seeds. We conducted the P content measurement of seeds and found that the P level of vpt1vpt3 double mutant seeds was nearly 30% higher than that of WT (Figure 2f). Thus, downregulation of VPT1 and VPT3 in leaves during the flowering stage would enhance P accumulation in seeds, which helps to store enough P source for seed germination.

Genetic control of VPT1 contributes to reduction of phosphorus contents in seeds
As described above, downregulation of VPT proteins resulted in more Pi allocated to reproductive organs. Therefore, overexpression of VPT1, the major vacuolar Pi influx transporter, may reduce Pi contents in the reproductive organ. To verify this hypothesis, we generated dexamethasone-inducible VPT1 overexpression lines (DiVPT1). After spraying dexamethasone regent on the surface of DiVPT1 leaves, we found that the expression of VPT1 was significantly upregulated in the leaves (Figure 3a). We then gathered leaves, xylem soup and floral organs for measuring Pi contents. The data shown in Figure 3b,c illustrated that induction of VPT1 dramatically upregulated Pi contents of leaves, while the Pi concentration in xylem soup was significantly downregulated. Meanwhile, the floral organ Pi content of VPT1 inducing seedlings was lower than that of WT and the un-induced seedlings (Figure 3d). Therefore, overexpression of VPT1 in leaves would systemically control the Pi distribution in plants.
As induction of VPT1 in leaves resulted in less xylem Pi, the P level in seeds may also be downregulated. Then, we cultured WT and DiVPT1 seedling lines for phenotyping. Firstly, we found that overexpressing VPT1 in leaves of DiVPT1 lines did not affect silique development and seed production. The length and seed number of the DiVPT1 siliques were at a comparable level as compared to WT (Figure 4). Moreover, the seed size and the weight of thousand seeds of DiVPT1 lines were also similar with WT (Figure 5a-c). However, the P contents of VPT1-induced DiVPT1 seedlings were more than 20% lower than that of WT (Figure 5d). We then analyzed the germination rates of the various seeds and found that the lower phosphorus containing seeds had a comparable germination rate when compared with WT seeds (Figures 5(e,f)). Thus, overexpression of VPT1 contributes to low P containing seeds without affecting the seed vigor.
In summary, this study explored the strategy of reducing P level in seeds. We firstly tested the Pi distribution pattern in plants during the reproductive stage, and we found that VPT1 and VPT3 genes, the major Pi transporters responsible for maintaining systemic Pi homeostasis, were regulated for monitoring Pi distribution among different tissues. The plants tend to suppress VPT1 and VPT3 for stimulating Pi allocation into reproductive organs rather than storage in leaves and thus resulted in high P seeds. Aimed at this case, we successfully downregulated the seed P content by site-specific overexpression of VPT1 with an inducible system (Figure 5g). Recent studies suggested that  reducing Pi allocated in grains would result in decreasing PA in seeds and enhanced grain filling for rice production 12,13 , which would contribute to a more environmentally friendly and sustainable agriculture. Therefore, maintaining a low content of Pi in the reproductive organ is important in crops. Based on the data in the study here, we believe that genetically engineering VPT-type proteins in crops such as rice and maize may be a promising strategy to generate low-P containing grains. Moreover, our study strengthens the notion that transporters represent the ideal control points and genetic control of the important nutrient transporters will surely benefit the sustainable agriculture in the future.

Plant materials and growth conditions
Arabidopsis Col-0 seedlings were used in this research. The vpt1-1 (SAIL_96_H01), T-DNA insertion mutant, was purchased from the Arabidopsis stock center. The hydroponic culture solution was prepared as the recipes in the previous study 14 . For the dexamethasone treatment, 10 µM dexamethasone solutions were sprayed on leaves of the flowering DiVPT1 seedling lines. We treated DiVPT1 seedlings with dexamethasone every 5 d in the early flowering stage till to seed mature stage. vpt1 vpt3 double mutant used in this study is vpt1-1 vpt3 11 . All the seedling lines were grown under a long day cycle (16-h light/8-h dark) at 22°C.

Vector construction and plant transformation
For the dexamethasone-induced VPT1 vector, the stop codon omitted CDS of VPT1 was amplified from wild type cDNA and cloned into a modified binary vector pCAMBIA-1300 with a dexamethasone-inducing promoter. For the GUS assay vectors, the native promoters of VPT1 and VPT3 were fused with GUS and cloned into a modified binary vector pCAMBIA-1300. The primers used for plasmids constructions are listed in Supplementary Table S1. The Agrobacterium tumefaciens stain carrying indicated construct (GV3101-pSoup19) was used to transform Arabidopsis Col-0 seedlings.

GUS staining
The GUS staining was performed as previously described but with small modifications. Briefly, the tissues of stable transgenic seedlings were fixed by incubation in 80% acetone for 15 min and then washed three times with the washing buffer (50 mM NaPO 4 pH 7.2, 1 mM K 3 Fe[CN] 6 , and 1 mM K 4 Fe[CN] 6 ). Then, the plant tissues were submerged in the staining buffer (50 mM NaPO 4 pH 7.2, 10 mM Na 2 EDTA, 0.1% Triton X-100, 1 mM K 3 Fe[CN] 6 , and 1 mM K 4 Fe[CN] 6 , 2 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide) and vacuumed for 20 min. Finally, the tissues with staining buffer were incubated for 8 h in the dark at 37°C. The stained tissues were decolorized with 70% ethanol and photographed.

RNA isolation and reverse transcription
The plant tissues were immersed in liquid nitrogen and grounded. Total RNA of different samples were extracted by Trizol reagent (Invitrogen). Two-microgram RNA of each sample was used for synthesizing the first-strand cDNA with oligo(dT) primers. qPCR was performed by QuantiFast SYBR Green PCR Kit (Qiagen) on a CFX Connect Real-Time System (Bio-Rad). The specific primer pairs used in qPCR are listed in Table S1.

Xylem sap collection
The flowering WT and stable transgenic DiVPT1 lines were cultured in the hydroponic solution with sufficient Pi concentration (130 µM). After spraying dexamethasone, the plants were decapitated at the bottom of the stem. The first drop of the xylem sap was discarded, and the subsequent sap was collected every 15 min for 60 min. High humidity (relative humidity is more than 70%) of the plant culture environment is necessary during this experiment. The xylem sap was centrifuged at 12,000 rpm for removing debris, and 20 µl supernatant of each sample was used for Pi concentration measurement or stored at 4°C for later assays.

Pi content assay
Different tissues of various genotype seedlings grown in the hydroponic culture were collected and washed three times in distilled water. We used 50 mg of each tissue samples or 20 µl xylem sap from various genotype seedlings for Pi content measurement following the ascorbate-molybdate antimony method as previously described 4 .

Determination of the seed P contents
Phytic acid (PA, or InsP6) is the major form of P in seeds 6-8 , and we therefore measured the seed P content through nitric acid digestion method. Briefly, 20 mg of each seed sample from different genotypes were digested in the liquid nitric acid, so that all the organic phosphorus in seeds were transformed to be inorganic phosphorus (Pi). Then, the digested liquid samples were gathered for Pi measurement through ascorbatemolybdate antimony protocol as previously described.

Statistical analysis
The data in this study are average values from at least three independent experiments, and the values were subjected to statistical analysis through one-way analysis of variance (ANOVA) followed by Student's t-test or Tukey's honestly significant difference test.

Accession numbers
Sequence data for genes presented in the current study can be found in the Arabidopsis Genome Initiative of GenBank/EMBL database under the following accession numbers: VPT1 (AT1G63010), VPT3 (AT4G22990), UBQ10 (AT4G05320), FT (AT1G65480).